Objective To study role of bone marrow mesenehyme stem cells in tumor formation.Methods Nude mice (n = 18) were randomly divided into two groups.Mice in the control group were subcutaneously injected with human embryonic MSCs, and F6 cells were injected into the nine mice of the experimental group.Three mice were sacrificed to remove tumor tissue, which was then prepared for real-time BT-PCR detection at 4 (F6-4), 6 (F6-6) and 7 (F6-7) weeks, respectively.Human lung cancer tissue samples and adjacent non-malignant lung tissue samples were collected from 4 lung cancer patients.The difference between gene expression of F6 cells and MSCs was distinguished by fluorescent differential display (FDD) and verificated by PCR and the western blot analysis. Real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-BT-PCR) was used to detect gene expression in tumor tissues after tumorigenesis in nude mice.A new tumor cell line, denominated F6, was established from mutated human embryonic bone marrow mesenchymal stem cells (MSCs) which were induced by the GMCSF and IL-4 in vitro.Results FDD analysis confirmed that cyelin Ⅰ was positively up-regulated in F6 cells compared with MSCs. Similar results were obtained by PCR and western blot.The cyclin Ⅰ gene expression levels in MSCs, F6, F6-4, F6-6 and 176-7 were significantly different(F=12.29 ,P ＜ 0.01).The cyclin Ⅰ gene expression level in F6(4.49±0.40) was 112 folds higher than those of MSCs(0.04±0.02,P＜0.01).Expression levels in F6-4 , F6-6 and F6-7 tissue were 1.82±0.80,3.30±0.43,3.68±1.67 which were significantly higher than that in MSCs (P＜0.05 or ＜0.01).The expression of cyclin Ⅰ increased significantly alone with the accreting volume of tumor.The expression levels of cyelin Ⅰ in human lung cancer tissues in four patients were 0.15±0.02, 0.58±0.23, 4.82±1.12, 1.21±0.60,respectively, and were significantly higher than that in adjacent non-malignant lung tissues (0.04±0.02,0.09±0.04,0.94±0.74,0.15±0.08) (F=12.39,P＜0.01).The protein expression level of cyclin Ⅰ in F6 cells was 0.32±0.08, which was 3.6-fold higher than that in MSCs (0.09±0.06, t=3.86,P＜0.05).Conclusions The expression level of cyclin Ⅰ in tumor tissue is higher during the entire course of tumorigenesis.Cyclin Ⅰ might be one of the factors playing important roles during tumorigenesis,especially in MSCs mutation.

Objective To monitor and analyze the distribution of pathogenic bacteria from CSF and its drug resistance change in Yangzhou area during 2011-2015 ,so as to provide the latest evidence for clinical rational use of antibacterial drugs .Methods The VITEK 2 automatic microbiological instrument was applied to identify bacteria and conduct the drug susceptibility test .The distri‐bution and drug susceptibility situation of isolated pathogenic bacteria were analyzed by using the WHONET 5 .6 software . Results In 2074 CSF bacterial culture from 2011 to 2015 ,74 strains(3 .57% ) of pathogenic bacteria were isolated ,in which the top three were Acinetobacter baumanni(21/74 ,28 .38% ) ,Klebsiella pneumonia (13/74 ,17 .57% )and Staphylococcus epidermis(12/74 , 16 .22% ) .The resistance rate of acinetobacter baumanni toantibacterial drugs was extremely serious ,showing muti‐drug or pan‐drug resistant phenomena .Conclusion Regular monitoring and analyzing the species and change of drug resistance in pathogenic bacteria isolated from CSF have an important significance to guide clinic to rationally use antibacterial drugs .

Objective To investigate the clinical distribution and antimicrobial resistance characteristics of carbapenem-resistant Acinetobacterbaumannii (CRAB).Methods CRAB isolated from all inpatients in a hospital in January-December 2015 were performed retrospective analysis,antimicrobial susceptibility testing result was analyzed.Results A total of 721 AB strains were detected,231 (32.04%)of which were CRAB,isolation rates of CRAB in quarter 1-4 were 48.99% (73/149),41.98%(68/162),27.39%(63/230),and 15.00%(27/180) respectively,which showed a decreased trend (P<0.001).CRAB mainly came from sputum specimen(n=140, 60.61%),followed by secretion of wound(n=33,14.28%)and urine specimen(n=24,10.39%).CRAB mainly distributed in intensive care unit,accounting for 43.72%(n=101),following by department of neurosurgery(n=37,16.02%),and burn/plastic surgery (n=22,9.52%).Resistance rates of CRAB to ampicillin/sulbactam, gentamicin,levofloxacin, and ciprofloxacin were 85.28% -90.48%,resistance rate to tobramycin was low (19.48%),no strains were found to be resistant to polymyxin B.Conclusion Antimicrobial resistance of CRAB is serious,it is necessary to focus on management of key departments,take scientific prevention and control measures, so as to effectively reduce the incidence of healthcare-associated infection.

Objective To study the tumorigenesis mechanism in bone marrow mesenchyme stem cells (MSC). Methods The bone marrow MSC could be induced into turnout (F6 cells) in vitro. The difference between gene expression of F6 cells and MSC was distinguished by fluorescent differential display (FDD). Verification of the result was detected by Real time RT-PCR and Western blotting, and immunocytochemistry. Results FDD analysis confirmed that Nucleostemin (NS) was positively up-regulated in F6 cells compared with MSC. Similar results were obtained by PCR and Western blotting. The NS gene expression levels in MSC, F6, 176-4, F6-6 and F6-7 were significantly different(F =160, P <0.05). The NS gene expression level in F6 (0.0372±0.0019) was 18 folds higher than those of MSC(0.0021±0.0002,P <0.05). Expression levels in F6-4, F6-6 and F6-7 tissue were 0.0504±0.0083, 0.0995±0.0026 and 0.0614±0.0036, and were significantly higher than that in MSC(P <0.05). The expression of NS increased significantly with the accreting volume of turnour, and high-level protein expression of NS was confirmed by Western blotting and immunocytochemistry. Conclusion The expression level of NS might be one of the factors playing important roles during turnour genesis, especially in MSC mutation.

Objective To establish an isolation method for solid GTC in peripheral blood using EpCAM antibody-linked nanobeads and evaluate the sensitivity of the method and its application significance. Methods Five, ten, twenty, fifty and one hundred MCF7 (breast cancer), KYSE70 (esophageal cancer), BxPC-3 (pancreatic cancer) and 9811P (stomach cancer) cells were added into 7. 5 ml erythrocyte lysed peripheral blood obtained from healthy volunteers respectively. EpCAM antibodylinked nanobeads were used to enrich cancer cells. The recovery rates of the in vitro added cancer cells were evaluated by fluorescence microscopy. Then, the untreated thirty cases of esophageal cancer (six cases at stage Ⅰ and Ⅱ, twenty-four cases at stage Ⅲ and Ⅳ), thirty-five cases of breast cancer (fifteen cases at stage Ⅰ and Ⅱ , twenty cases at stage Ⅲ and Ⅳ), thirty cases of pancreatic cancer (five cases at stage Ⅰ and Ⅱ , twenty-five cases at stage Ⅲ and Ⅳ), thirty-three gastric cancer (thirteen cases for stage Ⅰ and Ⅱ ,twenty cases at stage Ⅲ and Ⅳ) were enrolled to enrich the peripheral blood CTC. Thirty healthy volunteers and thirty gastritis patients served as two groups of control. Meanwhile the enriched CTC was identified by IF and HE staining. FISH was used to analyze the copy number of chromosome 8 and chromosome 20 in two hundred esophageal cancer, breast cancer, pancreatic caner and gastric cancer CTC. Results After DAPI staining and mixing with 7.5 ml peripheral blood from healthy donors, the average cell recovery rates of KYSE70, MCF7, BxPC-3 and 9811P cells evaluated under fluorescence microscope were 87%, 87%, 86% and 88% (within group), and the recovery rates of 5 gradient dilution levels were 88%, 85%, 87%, 88% and 87% (intergroup). With a high sensitivity, this method was able to isolate one cancer cell in 107 white blood cells of peripheral blood. The positive rates of more than 2 CTC in the peripheral blood detected by this method were 50% (15/30) of esophageal cancer, 63% (22/35) of breast cancer, 70% (21/30) of pancreatic cancer and 61% (20/33) gastric cancer patients respectively,but no CTC was detected in the peripheral blood of healthy volunteers and gastritis patients (P = 0. 000).The aneusomy of chromosome 8 and chromosome 20 were found in 80% esophageal cancer, 75% breast cancer, 65% pancreatic cancer and 59% gastric cancer. Conclusions The CTC isolation technique with EpCAM antibody-linked nanobeads is sensitive and accurate. The aneusomy of chromosome 8 and 20 is frequent in CTC from esophageal cancer, breast cancer, pancreatic cancer and gastric cancer.

Objective To construct a lentiviral vector containing mir-7-3 gene and green fluorescent protein (GFP) gene,and to detect the expression of mir-7-3 gene in U251 cells.Methods The fragments containing all the mir-7-3 gene were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP,which was transfected together with the packaging plasmids into 293T cells by CaC12.The supernatant was collected,concentrated,identified,and was transfected to U251 cells of gliomas.Fluorescent microscopy was used to observe the fluorescence in the 293T cell,and real time RT-PCR was used to examine the relative contents of mir-7-3 in U251 cells.Results Electrophores was shown that the sequence of the RT-PCR product was consistent with the data of mir-7-3 by DNA sequence analysis,indicating that the mir-7-3 gene was successfully cloned,and strong green fluorescence was observed by fluorescent microscopy.The supernatant of lentivirus-transfected 293T cells effectively infected U251 cells and the relative content of mir-7-3 was observed in the transfected U251 cells.Conclusion It is concluded that the lentiviral vector containing mir-7-3 gene was constructed successfully,which provides a basis for further study of mir-7-3 function.

Objective In this study, we constructed a plasmid, specifically expressed SUMO protein, and to study the expression level of anti-SUMO antibody in the serum of patients with primary biliary cholangitis, systemic lupus erythematosus, rheumatoid arthritis, Connective tissue disease, sjogren′s syndrome which were typical of autoimmune diseases.And to investigate whether anti-SUMO antibodies can be used as specific serum markers of PBC.Methods Plasmids containing SUMO1, SUMO2 and SUMO3 fragments were prepared by PCR and ET cloning and introduced into E.coli for protein induction and purification, respectively.Dot blot was used to preliminarily screen anti-SUMO antibody-positive specimens from serum samples of PBC patients and were verified by western blot to obtain positive reference serum.Through the establishment of the optimal anti-SUMO antibody ELISA diagnostic system, the positive rates of three subtypes of anti-SUMO antibody in PBC, SLE, SS, RA and CTD were detected, and their differences were analyzed by chi-square test.ResultsThe anti-SUMO antibody label has a specificity of up to 99％in PBC and a sensitivity of around 86％.After chi-square test analysis, the positive detection rate of anti-SUMO antibody in PBC was higher than that of nonPBC autoimmune disease and healthy controls (P＜0.01).There was no significant difference in the expression of anti-SUMO antibody in other autoimmune disease populations (non-PBC autoimmune diseases) (P＞0.05).Conclusion The highly specific anti-SUMO antibody is expected to become a novel antibody for diagnosis of PBC, which is of great significance for improving the clinical diagnosis efficiency of PBC.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9),a cluster of regularly spaced short palindromic repeats,is a natural immune defense system for bacteria and archaea to identify themselves and exogenous invading DNA fragments,protecting them from viruses.In recent years,CRISPR/Cas9 has become a revolutionary gene editing tool.Its specific targeted spot-cutting ability also plays an important role in nucleic acid detection,bacterial typing,etc.,and has shown great application potential in the field of medical testing.Based on the latest researches,this paper reviews the progress of CRISPR/Cas9 application in the new techniques of nucleic acid detection,pathogen typing and bacterial evolution in laboratory medicine,and also summarizes the application prospect of CRISPR technology in the field of laboratory medicine.

Objective To construct a lentiviral vector containing mir-7-3 gene and study the function of mir-7-3 gene and its role in glioma gene therapy.Methods The plasmid Lenti-GFP-mir-7-3 and packaging plasmids pRsv-REV,pMDlg-pRRE and pMD2G were co-transfected into the human embryonic kidney epithelial cell line 293T cells,and then,recombinant lentiviral FIV-CMV-GFP-mir-7-3 carried mir-7-3 gene and GFP gene was obtained; fluorescence expression of 293T cells was observed under fluorescence microscope.The supematant was collected,concentrated and identified,and then,it was used to transfect into the U251 glioma cells (positive transfection group); and blank control group (cells transfected with empty vector) and negative control group (parental cells) were also employed.Real time-PCR was used to examine the relative contents of mir-7-3 in U251 cells; Westem blotting was employed to detect the epidermal growth factor (EGFR) and serine/threonine kinase (AKT2) protein expressions; cell cycle was analyzed by flow cytometry and cell proliferative activities were measured by MTT method.Results Recombinant lentiviral FIV-CMV-GFP-mir-7-3 carried mir-7-3 gene and GFP gene was successfully constructed in 293T cells; electrophores showed that the sequence of RT-PCR product was consistent with the data ofmir-7-3 by DNA sequence analysis,indicating that the mir-7-3 gene was successfully cloned; strong green fluorwscence was observed by fluorwscent microscopy.The supernatant of lentivirus-transfected 293T cells was effectively infected into U251 cells and the relative content of mir-7-3 could be observed in the transfected U251 cells.As compared with those in the parental cells and the cells transfected with empty vector,the protein expressions of EGFR and AKT2 in the transfected group decreased significantly,reaching 38％ and 42％,respectively (P＜0.05).As compared with those in the parental cells and the cells transfected with empty vector,the cells at G0/G1 phase increased,the S phase fiaction was lower and the survival rates dramatically dropped in the mir-7-3 transfected cells 3,4,5 and 6 d after implanation.Conclusions The lentiviral vector containing mir-7-3 gene was constructed successfully.Mir-7-3 could specifically suppress EGFR and AKT2 expressions,induce gene silencing,inhibit cell growth,indicating that this way should be a new strategy in glioma gene therapy.

The application of Raman spectroscopy in the field of laboratory medicine is making continuous progress and development. The biosensor platform based on Raman spectroscopy provides a new means for accurate molecular diagnosis of diseases. In particular, as a fast and non-destructive detection method, surface-enhanced Raman scattering has the advantages of simple sample preparation, little interference from water and real-time detection, and shows great application potential in the field of medical examination. At the same time, with the integration of SERS and other technologies, including electrochemistry, new nano-materials, microfluidic, biochip, DNA nano-machine, artificial intelligence and machine learning, it will play a more and more important role in the field of medical laboratory. With the deepening of SERS research and the cross-integration between multiple disciplines, it will be widely used in biomedical detection and is expected to become an important technology platform for the next generation of precision diagnosis.